Method for producing tissue microarray blocks of cell cultures

ABSTRACT

A method and apparatus are provided for forming high-yield tissue microarray blocks capable of producing 1,000 or more replicate slides. In one embodiment, a plurality of donor tissue samples are minced and suspended in separate molten liquid carriers to form a plurality of separate two-phase mixtures of solid minced tissue and molten wax. Each of the two-phase mixtures is preferably formed into elongated columns and transferred into a separate well of a microarray block. A method and apparatus are provided for forming the elongated molten wax columns with minced tissue evenly distributed along the length of each column. The apparatus for forming the array includes an extrusion mechanism for transferring the columns of two-phase mixtures into the deep wells of the microarray block. The two-phase mixture may alternately be cooled and allowed to solidify before being transferred into the microarray block. An alternate embodiment is provided wherein cell cultures are utilized as starting material as opposed to solid donor tissue samples.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. nonprovisional application Ser.No. 12/156,700 filed Jun. 4, 2008, now U.S. Pat. No. 8,911,682, whichclaims the benefit of and priority from U.S. provisional applicationSer. No. 60/933,968 filed on Jun. 8, 2007, the entire content of each ofwhich is incorporated herein by this reference.

BACKGROUND AND BRIEF SUMMARY OF INVENTION

The present invention relates generally to tissue arrays used inhistology for medical and biological research as well as in medicaldiagnosis and treatment of disease.

The virtual explosion of knowledge in biotechnology in recent years hascaused an enormous demand for tissue microarray blocks. These microarrayblocks contain tissue samples that provide multiple on-slide tissuesthat are useful for a variety of purposes.

The prior art techniques for producing tissue microarray blockstypically involve transferring of solid core samples from a donor tissuesample into a blank recipient wax block. The thickness of these priorart solid core samples is determined by the thickness of the donortissue. As an example, if the donor tissue has a thickness of 2 mm, thesolid core sample transferred into a well of a prior art tissuemicroarray block will have that same 2 mm thickness. When sliced intosections for microscopy, that particular tissue sample can produce about200 slides. In a prior art tissue microarray block having a plurality ofwells containing different solid tissue samples with differingthicknesses, each well will produce a different number of “daughter”slides. This results in an inefficient technique, since the well withthe thinnest solid core sample will determine the ultimate number ofslides obtainable from the block. Therefore, in the prior art technique,the number of obtainable slides is limited, and there is also a waste ofdonor tissue.

There is clearly a dire need for a more efficient technique of producingtissue microarray blocks wherein each well of the block contains a solidtissue sample having a depth not determined by the thickness of thesolid donor tissue. In fact, for the purposes of immunohistologicdiagnosis, the College of American Pathologists has recommended multipleon-slide tissue controls for every patient (Commission On LaboratoryAccreditation, Laboratory Accreditation Program Anatomic PathologyChecklist, S. Sarewitz, MD, Editor, September 2007, pp. 45-46). Giventhe enormous demand for tissue microarrays (TMAs) that this wouldrepresent, this goal has not yet been realized, and a more efficientmethod for TMA production is required. The present invention providesfor more efficient TMA production and, in addition, achieves a“high-yield” tissue microarray block capable of producing many timesmore slides from a single block than the prior art!

The manner in which the present invention achieves these dramaticresults is elegant in its simplicity. Rather than using solid,rod-shaped coring samples, one embodiment of the present invention“minces” the solid donor tissue into fragments. The fragments areembedded in a liquefied carrier having a low melting point, such as wax.The solid, minced fragments are evenly distributed throughout the lengthof the molten, liquefied carrier, forming a two-phase (i.e. solid andliquid) mixture. The two-phase mixture may be formed into the shape of a“deep well” and transferred into a microarray block while the carrierremains molten. In another embodiment, the two-phase mixture is cooledto solidify the carrier prior to being transferred into a “deep well,”such as 17 mm long and 2 mm diameter, for example. By using this“mincing” technique together with forming the desired shape of a deepwell, both objectives described above are achieved along with reducedwaste of donor tissue. First, the thickness of the solid donor tissuetransferred to each well is not limited to the thickness of the originaldonor tissue. Second, and equally important, the thickness of the tissuesamples transferred into the wells of the block is uniform and of muchgreater thickness than the prior art. The result of the presentinvention is an efficient, cost effective, high quality and high yieldtissue microarray block capable of producing 1,000 to 2,000 replicateslides, 5 to 10 times more than the prior art!

Alternatively, rather than “mincing” solid donor tissue, homogenoustissue derived from cell culture may be used and, after appropriatefixation, suspended in a liquid carrier and transferred in liquid forminto a deep well. In a distinct version of the technique, the mixture ofcell culture material and carrier can be allowed to solidify within aduct of cross-sectional size and shape approximately equal to that ofthe deep well, and subsequently transferred in solid form into the deepwell by simple extrusion from said duct, via the simple application offorce by a plunger or other means. Both of these techniques differ fromprior art techniques using cell cultures to form TMAs, in that the priorart solidifies the mixture of cell culture and carrier (bycentrifugation, re-suspension in a carrier and solidification of themixture) and then takes core samples of the resulting solid material(Moskaluk and Stoler, “Agarose Mold Embedding of Cultured Cells forTissue Microarrays,” Diagnostic Molecular Pathology 11(4): 234-238,2002; also Montgomery et al, “A Novel Method for Making ‘Tissue’Microarrays From Small Numbers of Suspension Cells,” AppliedImmunohistochemistry & Molecular Morphology, 13(1): 80-84, 2005).

Another aspect of the present invention is a novel system for formingthe two-phase mixture of minced solid donor tissue into elongatedcolumns (17 mm long and 2 mm in diameter, for example) of a meltablecarrier such as wax. An elongated passageway is formed in a body ofthermally conductive, preferably transparent material. A source ofsufficient heat is applied to the passageway in order to maintain partor all the length of the passageway above the melting-point temperatureof the carrier substance. Paraffin wax is a good example of a suitablecarrier, but other substances can also be used. Molten carrier istransferred into the elongated passageway by one or more of severalmeans: (a) the pure carrier substance may be introduced in the moltenstate from a heated dispenser; (b) the pure carrier substance may beintroduced in the solid state, relying on the temperature of thepassageway to melt the carrier; (c) tissue samples may be embedded inthe carrier substance, which melts due to the temperature of thepassageway. Option (c) is often preferable in the case of minced solidtissue fragments. In any case, the minced solid donor tissue fragments,or other tissue constituents, such as obtained from cell cultures, areintroduced to the passageway along with the molten carrier substance,thus creating a two-phase mixture (suspension of biological material inliquid carrier). At the entrance of the passageway may exist acollecting receptacle or “hopper,” also heated, to facilitate themelting of carrier and the mixing of biological material in liquidcarrier. A duct originates in the hopper and intersects the passageway.With or without the use of a hopper, the liquid carrier and biologicalmaterial are inserted into the passageway. Some means of propelling thistwo-phase mixture through the passageway is implemented, such as: (a)pressing with a plunger that contacts the mixture and propels itsimilarly to the action of a syringe; (b) the application of hydraulicpressure to the passageway after sealing any side-ports such as theintersecting duct from a hopper; (c) gravity-driven flow of the mixture;(d) propulsion of the mixture by direct contact with an auger feederwithin the passageway. It is the liquid state of the carrier during thisstep that enables such a range of possible propulsion mechanisms, andalso in general enables the mixture to flow through a duct withcurvature, converging and/or diverging sections. During its travelthrough the passageway, the velocity and temperature of the mixture arecontrolled by the speed or pressure of the propulsion mechanism and, ifnecessary, the management of heat applied to the passageway, order toform the mixture into a dense column and to control the state ofliquidity of the carrier substance. Some degree of cooling, in order toincrease the viscosity of the liquid carrier, or even to solidify it,can be advantageous near the end of the passageway. The compressedtissue column is then extruded out of the elongated passageway into awell within a recipient block.

In the case where the biological material for the microarray comes froma solid human or animal tissue source, the mincing step must be done inorder to obtain a sufficiently pure tissue of interest, and withtechniques appropriate to the type of tissue and to the requiredorientation of tissue structures in the product slides and hence in themicroarray block. In summary, the process of mincing depends on the typeof tissue. In many cases, skilled dissection by hand is required, but inother cases mincing can be done by an automated fragmenting process suchas with sharp rotating blades. As used herein and in the claims, theterm “mincing” is used in a broad sense to include (a) simplefragmentation of a solid tissue sample that has been previously selectedfor its purity of tissue type, when purity of tissue type is sufficientfor the purposes of the resulting microarray analysis; (b) carefuldissection into fragments of a more precisely specified biological andphysical structure, when such precision is necessary in order that therequired biological and physical structures are present in the resultingmicroarray. Two illustrative examples are provided in the nextparagraph.

Careful dissection may be required to harvest the tissue of interest atvarious steps depending on the tissue chosen. Illustration is providedby way of two examples that demonstrate some of the range of precisionrequired for different tissue types. Colonic epithelium is harvestedfrom a colonic resection specimen, preferably one that is non-fixed. Thecolon is opened longitudinally, using sharp dissection (scalpel blade)to separate the mucosa from the underlying fibromuscular soft tissue.Sheets of colonic mucosa are obtained in this manner and are cut intosmall sections of appropriate size for placing in plastic histologycassettes (up to about 20 millimeters×10 millimeters×4 millimeters). Thesectioned mucosa is submitted in cassettes for routine histologicfixation and “processing” (dehydration and embedding in wax). Afterprocessing, the wax shroud is melted away, and the solid tissue isminced with a scalpel blade. Minced fragments should be on the order oftwo millimeters×two millimeters×two millimeters. The size of thefragments and their orientation can be important in some circumstances.For instance, skin fragments should have a long axis parallel to theplane of the dermal-epidermal junction, and this axis should be loadedparallel to the long axis of the “barrel” of the injector, such that thedermal-epidermal junction is ultimately visualized in cross-section onthe microscope slide.

A further aspect of the present invention is a novel apparatus forforming the recipient (blank, open-well) microarray blocks. Theapparatus provides an efficient system for forming the required array ofmuch deeper wells than the wells of the prior art. The apparatusincludes a means of easily varying the number and configuration of wellsby removable/configurable pins in a mold device. This is useful forcustomizing the size and shape of the array. However, it should be notedthat a casting technique is not the only way to produce the recipientmicroarray blocks. For example, an alternative and efficient methodwould be to use a drilling, reaming, and/or milling method for creatingdeep wells in an initially solid block of machinable wax, using a manualor computer numeric controlled (CNC) milling or drilling machine.

The prior art techniques of forming tissue arrays are summarized in thearticle by Eguiluz et al entitled “Multitissue array review: Achronological description of tissue array techniques, applications andprocedures” published in Pathology-Research and Practice 202 (2006) atpp. 561-568 and in U.S. Pat. Nos. 6,103,518 and 7,029,615.

As noted above, the first embodiment of the present invention differssignificantly from the prior art by using minced solid tissue fragmentsto form a two-phase mixture with a molten carrier such as wax. The priorart uses core samples of solid donor tissue. The mincing (andsuspension) technique inherently allows the use of more uniform lengthwells and deeper wells than the prior art with less waste of donortissue.

A primary object of the invention is to provide a high yield tissuemicroarray block wherein minced solid donor tissue fragments aresuspended in a meltable or molten carrier such as wax.

An object of a second embodiment of the invention is to constructmicroarrays where the source of biological material comes from anothersource such as cell culture.

A further object of the invention is to provide a tissue microarrayblock wherein each well has an increased depth not determined or limitedby the thickness of the donor tissue.

Another object of the invention is to provide a method and apparatus forforming elongated columns of a meltable carrier, such as wax, whereinsuspended fragments of minced solid donor tissue are distributed evenlyover the length of the column.

Another object is to provide an apparatus for forming a tissuemicroarray block having a plurality of wells wherein each well has adepth significantly greater than prior art wells.

Other objects and advantages will become apparent from the followingdescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustrations of a donor tissue sample in solidwax in a step of a typical prior art method of preparing a tissuemicroarray block;

FIG. 1B is a schematic illustration of taking core samples in a step ofa typical prior art method of preparing a tissue microarray block;

FIG. 1C is a schematic illustration of removing core samples in a stepof a typical prior art method of preparing a tissue microarray block;

FIG. 1D is a schematic illustration of a recipient block with wells in astep of a typical prior art method of preparing a tissue microarrayblock;

FIG. 1E is a schematic illustration of a recipient block with coresamples placed in the wells of the recipient block in a step of atypical prior art method of preparing a tissue microarray block;

FIG. 2A is a schematic illustration of a donor tissue sample in solidwax in a step of the method of preparing a tissue microarray blockaccording to the present invention;

FIG. 2B is a schematic illustration of the donor tissue sample of FIG.2A after melting away of the solid wax according to the presentinvention;

FIG. 2C is a schematic illustration of the donor tissue sample of FIG.2A after mincing according to the present invention;

FIG. 2D is a schematic illustration of the minced donor tissue sample ina suspension of liquid wax according to the present invention;

FIG. 2E is a schematic illustration of a high-yield tissue microarrayblock with the two-phase solid/liquid tissue suspension injected intothe wells of the recipient block according to the present invention;

FIG. 3 is a perspective view of an apparatus for forming the individualtwo-phase columns of solid minced tissue and molten carrier according tothe present invention;

FIG. 4 is a perspective view of the apparatus shown in FIG. 3 which alsoincludes the heating device and temperature sensor according to thepresent invention;

FIG. 5 is a reverse perspective view of the apparatus shown in FIGS. 3and 4, partially in section and which also includes a tamper accordingto the present invention;

FIG. 6A is a schematic showing the preliminary loading of the two-phaseminced tissue and wax mixture into a hopper;

FIG. 6B is a schematic illustrating how the tamper shown in FIG. 6A isloading the solid minced tissue and wax mixture into the elongatedpassageway shown;

FIG. 6C is a schematic illustrating how the solid minced tissue and waxmixture is transferred or extruded into a deep well of a tissuemicroarray block according to the present invention;

FIG. 7 is a perspective view of a casting apparatus used in conjunctionwith the present invention;

FIG. 8 is an exploded view of the apparatus of FIG. 7;

FIGS. 9 and 10 show an alternate apparatus according to the presentinvention utilized to simultaneously prepare and transfer multiplemixtures of minced fragments and molten wax into a plurality of wells ofa tissue microarray block according to the present invention; and

FIG. 11A is a schematic illustration of a homogenous tissue samplederived from a cell culture and used as a “starting material” in analternate embodiment of the invention utilizing a cell culture ratherthan a solid donor tissue;

FIG. 11B is a schematic illustration of an elongated column withdispersed cells of the sample in molten wax adjacent a deep well block,in an alternate embodiment of the invention utilizing a cell culturerather than a solid donor tissue;

FIG. 11C is a schematic illustration of the transfer of the elongatedcolumn with dispersed cells of the sample in molten wax into the well ofthe block, in an alternate embodiment of the invention utilizing a cellculture rather than a solid donor tissue;

FIG. 11D is a schematic illustration of the plunger or piston driventowards the well to fill the well with the dispersed cells of the samplein molten wax, in an alternate embodiment of the invention utilizing acell culture rather than a solid donor tissue;

FIG. 11E is a schematic illustration dispersed cells of the sample incooled wax in the well and the withdrawn plunger, in an alternateembodiment of the invention utilizing a cell culture rather than a soliddonor tissue.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 1A-1E and 2A-2E are concept sketches which compare and contrastthe methodologies of the prior art (FIGS. 1A-1E) and the presentinvention (FIGS. 2A-2E).

As shown in FIG. 1A, a sample of solid donor tissue 20 is shown as beingstored or embedded in a body of solid wax 10, usually formalin fixed.The donor tissue is typically irregular in shape, typically having aregion 21 of minimum thickness and a region 22 of maximum thickness.

As shown in FIG. 1B, and according to typical prior art methodology, aplurality of coring tubes 31-36 are forced downwardly through donortissue sample 20 in order to transfer portions of the donor sample intothe coring tubes or punches.

As shown in FIG. 1C, the coring tubes 31-36 are then removed from thesample donor tissue; FIG. C shows only the core tissue samples 31 a-36 aand does not show coring tubes 31-36. Each core tissue samples 31 a-36 acontains a different thickness of donor tissue. For example, core tissuesample 36 a contains a donor tissue sample 36 a having a thickness t₁.

As shown in FIG. 1D, a prior art recipient block 50 is typically formedof wax, having wells 51-55 with a depth of d₁. A common prior art depthis 2 mm to 4 mm.

As shown in FIG. 1E, the next step in the typical prior art system is totransfer the cylindrical tissue core samples 31 a-36 a into the wells51-56 of recipient block 50, a traditional tissue microarray block,usually made of paraffin wax.

As is known in the prior art, the upper surface 59 of block 50 is slicedto obtain “daughter slices” of each of the core tissue samples 31 a-36a. Since core sample 36 a as the smallest thickness t₁ of each of thesamples 31 a-36 a, it will determine or limit the number of tissuesamples that may be obtained from all of the wells 51-56 of recipientblock 50.

FIGS. 2A-2E illustrate conceptually the methodology of the presentinvention.

As shown in FIG. 2A, the same sample solid donor tissue 20, as shown inFIG. 1A, is stored in a body of solid wax 10 as is known in the art.

As shown in FIG. 2B, according to the present invention, the wax body 10has been melted and the tissue sample 20 has been separated from the waxbody 10.

As shown in FIG. 2C, the solid tissue sample 20 has been “minced” intohundreds or thousands of solid tissue fragments 120. The mincing can bedone by a variety of techniques known in the art, preferably by repeatedslicing with sharp blades to produce relatively uniform sized fragments120.

As shown in FIG. 2D, the minced fragments 120 are then transferred intopreferably elongated, horizontal columns of liquid or molten wax 121such as column 161. As described below, vertical columns may also beutilized. The fragments 120 are introduced into the elongated wax column161 in a fashion (described in greater detail below), preferably toensure that the fragments 120 are distributed evenly throughout thelength of elongated column 161.

As shown in FIG. 2E, according to the present invention, a high yieldtissue microarray block 150 is provided having a body 159 and aplurality of relatively deep wells 151-156. Each of the wells 151-156 ispreferably cylindrical and preferably approximately 15 mm in depth andhaving a cross sectional diameter of approximately 2 mm. Thesedimensions are by way of example. It is to be understood that the welldimensions may be varied from the example given.

The depth d₂ of each of the wells 151-156 is preferably 15 mm to 20 mmor approximately 5 to 10 times greater than the depth d₁ of prior arttissue microarray blocks. Since the tissue fragments 120 arecontinuously distributed over the length of elongated column 161 carriedin well 151, usable “daughter slides” (or tissue microarray sections)can be obtained throughout the entire depth d₂ of each of the elongatedcolumns, such as 161 and 162, carried in wells 151-156 of the presentinvention. It is also significant to note that the present inventionuses essentially all of the sample donor tissue 20 and avoids waste ofthe sample tissue that is inherent in the prior art core sampletechnique described above.

FIGS. 3-6 illustrate the preferred method and apparatus of the presentinvention for preparing a liquid suspension of minced tissue fragmentsin molten wax and then extruding the suspension into the blank wells ofthe novel tissue microarray block of the present invention. Theextrusion device is shown generally as 200 and includes a housing 212having an elongated passageway 211 which extends through housing 212from the front wall 212 a and through the rear wall 212 b. An elongatedplunger 214 is mounted such that it can slide in the elongatedpassageway 211. A hopper 215 is formed in the top surface 212 c ofhousing 212 and extends downwardly and communicates with elongatedpassageway 211. Housing 212 preferably has three layers, including atop, thermally conductive layer 212 d, a middle metallic layer 217, anda lower polycarbonate, insulating layer 221. Housing 212 is preferablypositioned adjacent microarray block 150. An exit nozzle 227 is a thinwalled tube that is carried in passageway 211 and forms an extension ofelongated passageway 211. Nozzle 227 reduces or prevents leakage of thetwo-phase mixture as the mixture is transferred into the wells 151-152as shown best in FIG. 6C.

Referring to FIGS. 6A-6C, minced tissue fragments 223 which have beenpreviously trimmed (and preferably formalin-fixed and embedded inparaffin) are added to the hopper 215. A heating element 218 (FIG. 4) inthermal contact with passageway 211 causes the wax (in the solid state)in which the tissue fragments are embedded to melt, creating a liquidsuspension of tissue. Additional solid or liquid wax (or other lowmelting point carrier) can be added to the hopper 215 as needed toprovide a sufficient amount of liquid carrier for management of flow andelimination of bubbles and/or voids. It is also possible to processtissue fragments such as 223 that are not initially embedded with wax orother low melting point carrier material; the desired liquid for thesuspension can be created fully by adding solid or liquid carrier to thehopper 215.

The two-phase (i.e. liquid carrier and solid tissue fragments) mixtureis then pressed downwardly into the elongated passageway 211 with atamper 216. The tamper 216 is then used to seal the passageway, whilethe plunger 214 is utilized to compact the tissue fragments and meltedcarrier into a continuous column 161 (FIG. 6C) by advancing the plunger214 from its position shown in FIG. 6B in the direction of arrow 299.

As shown best in FIG. 6C, the high yield tissue microarray block 150 asshown in FIG. 2E is positioned adjacent housing 212 with the wells151-156 oriented in a horizontal fashion and aligned in a fashion so atleast one of the wells 151-156 is aligned with elongated passageway 211.As shown in FIG. 6C, plunger 214 is extruding the two-phase mixture ofsolid minced tissue and liquid carrier through thin walled tube ornozzle 227 into deep well 152 as a two-phase column of predeterminedlength shown as 162 in FIG. 2E in its finished form. Also shown in FIG.6C is well 151 having previously been filled with a similar column 161comprising a two-phase mixture of tissue fragments and molten waxwherein the column 161 has a uniform distribution of solid minced tissueover its length.

Alternatively, the column 161 of predetermined length may be allowed tocool and solidify in passageway 211 prior to being transferred orextruded into a well of a microarray block.

The outer diameter of the elongated passageway 211 is approximately thesame size as the inner diameter of each of the wells 151-156 of tissuemicroarray block 150. The process is repeated to fill all of the wells151-156 in block 150. The block 150 is then “cured” by uniformly heatingthe entire tissue microarray block in an oven, for example, to asoftening temperature (typically, 45 degrees Celsius for 30 minutes) topromote bonding between the tissue/wax mixture in each well and thesurrounding wax block 150. It should be noted that this curing step maybe similarly advantageous to be applied in conjunction with any of themethods stated herein for producing tissue microarray blocks, as a finalstep after transfer of tissue/wax mixture into the wells of therecipient block.

As shown best in FIG. 4, a heater 218 capable of heating housing 212 toapproximately 65-80 degrees Celsius is embedded in housing 212 and is inthermal contact with elongated passageway 211. A temperature sensor 219is also embedded in housing 212 and monitors operating temperatures ofthe device. A controller 220 is provided which may be either a voltagecontroller or feedback process controller utilizing the temperaturesensor 219 as an input to control the output of heater 218 in order tocontrol the temperature of material within elongated passageway 211.

FIGS. 7 and 8 illustrate the apparatus preferably utilized for formingthe paraffin blocks (such as 150 in FIG. 2E) with preformed deep wellsin a grid pattern array for use in conjunction with the presentinvention. The casting apparatus is shown generally as 300 in FIG. 7 andis shown in an exploded view in FIG. 8. As shown in FIG. 8, a modifieddeep histology block mold 301 is illustrated. The floor 301 a has beenmodified to create an opening to receive the pin assembly 303 describedbelow. Mold 301 includes upwardly oriented wings 301 b and 301 c on theshort sides to enable release of the wax block (not shown in FIG. 8 forclarity), by inverting the mold 301 and tapping the wings 301 b and 301c on a hard surface.

An ejector plate 302 is provided which is an elongated rectangular platewith an array of holes 302 a formed therein. The purpose of ejectorplate 302 is to separate the cast block (not shown) and mold 301 fromthe pin array assembly 303. Each pin of the pin array assembly 303 apreferably has a length of between 15 mm and 20 mm and a diameter ofpreferably 2 mm to 3 mm (as noted above). It is understood that the pinscould be larger or smaller and they could have a non-circular shape. Thepins form the deep wells of the finished block as shown as wells 151-156in FIG. 2E above.

A bridge assembly 304 is provided and cooperates with the ejector plate302 to facilitate the separation of the formed wax block (not shown inFIG. 8) from the pin assembly 303 a. The bridge assembly 304 includesfirst and second upstanding end pieces 304 a and 304 b. The end pieces304 a and 304 b are recessed as shown at 304 c and 304 d to receive theends 302 b and 302 c of ejector plate 302.

It is to be understood that various techniques may be utilized to formthe above-described two-phase mixture of minced tissue fragments andmolten wax into an elongated, preferably cylindrical column.

Molten wax 305 (FIG. 7) is poured through a slotted cover 306 into mold301 (FIG. 7).

FIGS. 9 and 10 illustrate an alternate embodiment of the apparatus andmethod shown in FIGS. 3-6. Whereas the embodiment shown in FIGS. 3-6 anddescribed above produces single, two-phase horizontal columns, theembodiment shown in FIGS. 9 and 10 may be utilized to produce multipletwo-phase mixtures of tissue fragments and molten wax simultaneously.

As shown in FIG. 9, a device shown generally as 400 includes a housing401 in which a plurality of generally circular hoppers 411-418 isformed. Each of the hoppers 411-418 is adapted to receive minced tissuefragments along with wax either in liquid or solid form. As shown inFIG. 10, each of the hoppers 411-414 has a downwardly extendingelongated passageway 411 a-414 a extending downwardly through the bottomsurface 402 of body 401 forming a manifold.

As shown in FIG. 10, a deep well block 450 (made of wax) according tothe present invention having vertical or slightly inclined wells 451-454is positioned adjacent to and immediately below the lower surface 402 ofhousing 401. The deep wells 451-454 are also aligned with the lower endsof elongated passageways 411 a-414 a. In this fashion, multipletwo-phased samples can be formed and transferred simultaneously into aplurality of wells of the novel deep well tissue block of the presentinvention. As shown in FIGS. 9 and 10, eight wells may be filledsimultaneously, only four of which (411-414) are visible in FIG. 10.Each well 411-414 has a continuous distribution of minced tissue (orcultured cells) from the bottom to the top of each well.

FIGS. 9 and 10 are schematic illustrations which do not include heatingelements and temperature sensing elements such as shown and describedabove for the sake of brevity.

FIGS. 11A-11E are “concept” sketches illustrating the alternateembodiment of the invention wherein a homogenous tissue sample 523 isderived from a cell culture and is used as a “starting material” ratherthan a solid donor tissue sample 20 as shown in FIGS. 1A and 2A anddescribed above. As shown in FIG. 11B, the tissue sample 523 has beenscraped or otherwise removed from the container in which it has beencultured (e.g. plastic flask or petri dish) or in which it has beendensified by centrifugation (e.g. centrifuge tube) and dispersed into apreferably horizontally disposed column 561 of molten carrier 591 suchas wax. Column 561 is held in a cylindrical duct or chamber (not shownin FIG. 11 for clarity), such as 211 in FIG. 6A. As used herein and inthe claims, the word “cylindrical” is defined in a broad sense toinclude elongated shapes with circular, oval, elliptical, rectangularand triangular cross-sections. The elongated column 561 is formedadjacent a deep well block 550 having a body 559 and a plurality of deepwells, such as deep well 551. As shown in FIG. 11C, a piston orextrusion rod 514 is actuated and is caused to move in the direction ofarrow 599 to transfer the elongated column 561 with dispersed cells ofsample 523 in molten wax 591 directly into the deep well 551 of block550. Appropriate measures are taken to prevent spillage or leakage ofthe molten column 561 as the transfer illustrated in FIG. 11C is takingplace. For example, deep well 551 may have an extension (not shown)formed around its outer lip 551 a to prevent leakage. Furthermore, themolten column 561 may be somewhat cooled prior to transfer to form aviscous two-phase mixture (or a single phase solid) in order to reducespillage or leakage. As shown in FIG. 11D, the plunger or extrusionpiston 514 has been driven in the direction of arrow 599 toward deepwell 551 and has completely filled deep well 551 with the dispersedcells of sample 523 carried in molten wax 591. As shown in FIG. 11E,molten wax 591 has been allowed to cool and solidify in well 551 priorto the withdrawal of the plunger 514. As shown in FIG. 11E, deep well551 has been filled with a continuous distribution of cells from sample523 throughout the length of deep well 551.

The foregoing description of the invention has been presented forpurposes of illustration and description and is not intended to beexhaustive or to limit the invention to the precise form disclosed.Modifications and variations are possible in light of the aboveteaching. The embodiments were chosen and described to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best use the invention in variousembodiments and with various modifications suited to the particular usecontemplated. The scope of the invention is to be defined by thefollowing claims.

1-22. (canceled)
 23. A method for use with a plurality of donor cellculture samples and a paraffin recipient block provided with a pluralityof wells to form a tissue microarray block, comprising the steps of:suspending each of the plurality of donor cell culture samples in aseparate liquid carrier to form a mixture of cell culture and liquidcarrier for each of the plurality of donor cell culture samples,compacting each of said mixtures of cell culture and liquid carrier intoa chamber to take the form of a column for use in a tissue microarrayblock, and transferring each of said columns from the chamber into atleast one of said plurality of wells of said paraffin recipient block tocreate the tissue microarray block, the length of each of said columnsbeing independent of the size of the respective donor cell culturesample. 24-29. (canceled)
 30. The method of claim 23 wherein saidtransferring step includes transferring the carrier in each of saidcolumns in a liquid state into the recipient block.
 31. The method ofclaim 23 further comprising cooling the carrier in each of said columnsprior to the transferring step so as to transform the carrier from aliquid state to a solid state.
 32. The method of claim 23 wherein eachsaid separate liquid carrier is a molten wax.
 33. The method of claim 32wherein said chamber is an elongated passageway and wherein saidcompacting step includes causing each of said mixtures of cell cultureand molten wax to separately pass into the elongated passageway to forma separate column.
 34. The method of claim 32 wherein each of saidplurality of donor cell culture samples is disposed in a separate waxand wherein said suspending step includes heating each separate wax toform said respective molten wax.
 35. The method of claim 23 wherein saidcompacting step includes pressing each of said mixtures of cell cultureand molten wax into said chamber.
 36. The method of claim 33 whereinsaid compacting step includes cooling each of said mixtures of cellculture and molten wax in said elongated passageway.
 37. The method ofclaim 33 wherein said suspending step includes embedding each pluralityof cell culture samples in a separate wax.
 38. The method of claim 23where said transferring step includes extruding each of said columnsfrom a passageway.
 39. The method of claim 23 wherein said compactingstep includes utilizing a plunger to compact each of said mixtures inthe chamber.
 40. The method of claim 23 wherein said transferring stepincludes causing each of said columns to flow into at least one of saidplurality of wells.
 41. A method for use with a donor cell culturesample and a paraffin recipient block provided with a plurality of wellsto form a tissue microarray block, comprising the steps of: suspendingthe donor cell culture sample in a liquid carrier to form a mixture ofcell culture and liquid carrier, compacting the mixture of cell cultureand liquid carrier into a chamber to take the form of a column for usein a tissue microarray block, and transferring the column from thechamber into at least one of the plurality of wells of the paraffinrecipient block to create the tissue microarray block.
 42. The method ofclaim 41 wherein the transferring step includes transferring the carrierin the chamber in a liquid state into the recipient block.
 43. Themethod of claim 41 further comprising cooling the carrier in the chamberprior to the transferring step so as to transform the carrier from aliquid state to a solid state.
 44. A method for use with a donor cellculture sample and a paraffin recipient block having a plurality ofwells to form a tissue microarray block, comprising the steps of:suspending the donor cell culture sample in a liquid carrier to form amixture of cell culture and liquid carrier, compacting the mixture ofcell culture and liquid carrier into a chamber to take the form of anelongate segment for use in a tissue microarray block, and transferringthe elongate segment from the chamber into at least one of the pluralityof wells of the paraffin recipient block to create the tissue microarrayblock.
 45. The method of claim 44 wherein the transferring step includestransferring the carrier in the elongate segment in a liquid state intothe recipient block.
 46. The method of claim 44 further comprisingcooling the carrier in the elongate segment prior to the transferringstep so as to transform the carrier from a liquid state to a solidstate.